Abstract

Cyclic nucleotides regulate axonal responses to a number of guidance cues through unknown molecular events. We report here that Drosophila nervy, a member of the myeloid translocation gene family of A kinase anchoring proteins (AKAPs), regulates repulsive axon guidance by linking the cyclic adenosine monophosphate (cAMP)–dependent protein kinase (PKA) to the Semaphorin 1a (Sema-1a) receptor Plexin A (PlexA). Nervy and PKA antagonize Sema-1a-PlexA–mediated repulsion, and the AKAP binding region of Nervy is critical for this effect. Thus, Nervy couples cAMP-PKA signaling to PlexA to regulate Sema-1a–mediated axonal repulsion, revealing a simple molecular mechanism that allows growing axons to integrate inputs from multiple guidance cues.

Many semaphorin proteins, including the Drosophila transmembrane semaphorin Sema-1a, are axonal repellents that signal through transmembrane plexin proteins (1). We identified nervy, a Drosophila homolog of the mammalian MTG8 proto-oncogene, in a yeast interaction screen as a Plexin A–interacting protein (fig. S1). Nervy belongs to a phylogenetically conserved protein family consisting of Drosophila nervy and three mammalian proteins: myeloid translocation gene (MTG) 8, MTG16, and MTGR1 (2–4) (Fig. 1A). Mammalian MTG proteins are tumorigenic (2–4) (fig. S1), but the cellular processes they control are poorly defined. MTG8 and MTG16 were recently found to bind the regulatory subunit of type II cAMP-dependent protein kinase (PKA RIIα) and function as PKA (or A kinase) anchoring proteins (AKAPs) in lymphocytes (3, 4). AKAPs position type II PKA at defined locations to allow for the spatially and temporally specific phosphorylation of target proteins in response to local increases in cAMP (5). Drosophila nervy shows considerable amino acid conservation with MTG proteins within a consensus PKA RII binding site (Fig. 1B) (4), suggesting that nervy may be an AKAP that couples cAMP-PKA signaling to semaphorin-plexin-mediated axon guidance. Cyclic nucleotides, including cAMP, are known to alter how axons respond to guidance cues, including semaphorins (6–9), but a direct molecular link coupling cAMP-PKA signaling to specific guidance cue signaling pathways has remained elusive.

Nervy, like PlexA (10, 11), is highly expressed in the Drosophila embryonic central nervous system (CNS), including in motor neurons (Fig. 1C) (2) and their axons (Fig. 1, C1 and C2). An antibody to a conserved region of mammalian MTG proteins also identified Drosophila nervy (fig. S2D) within CNS and motor axons (Fig. 1D). Immunoprecipitation of hemagglutinin (HA) epitope–tagged neuronal PlexA from Drosophila embryonic lysates revealed associated nervy (Fig. 1F), and neuronal HA-PlexA was detected in immunoprecipitates of nervy (fig. S2A), which suggests that nervy and PlexA interact in neurons. Nervy also immunoprecipitated with PKA RII in Drosophila embryos (fig. S2A), and an epitope (Myc) tagged neuronal nervy immunoprecipitated with Drosophila PKA RII (Fig. 1G), which indicates that nervy is a neuronal AKAP.

We then generated two nervy loss-of-function (LOF) mutants, nervy PDFKG1 and nervy PDFKG38 (fig. S2, B to E) and found that they exhibited highly penetrant axon guidance phenotypes consistent with increased axonal repulsion. Motor axons within the intersegmental nerve b (ISNb) pathway require Sema-1a-PlexA repulsive signaling to selectively defasciculate from the intersegmental nerve (ISN) and normally innervate muscles 6/7 and 12/13 (Fig. 2, A to C; Fig. 3, A and H; table S1) (10–13). In nervy LOF mutants, motor axons within the ISNb pathway often exited the ISN and ISNb in abnormal locations, were excessively defasciculated, and projected incorrectly within the ventral musculature (Fig. 2, D and F; Fig. 3, A and B; table S1). Motor axons within other pathways such as segmental nerve a (SNa) were also abnormally defasciculated in nervy LOF mutants and projected to inappropriate areas (Fig. 2F; Fig. 3, A and B; fig. S3B; table S1). CNS projections were also abnormal in nervy LOF mutants. In wild-type embryos, three evenly spaced and uniformly thick longitudinal axon bundles were detected on each side of the CNS with an antibody to fasciclin II (FasII) 1D4 (Fig. 2, B and C; Fig. 3A; table S1) (11, 12)). In nervy LOF mutants, axons within the third, most lateral, longitudinal bundle were less tightly fasciculated and often extended away from the CNS in inappropriate bundles (Fig. 2, E and F; Fig. 3, A and B; table S1). A full-length nervy transgene expressed in all neurons rescued these axon guidance defects in nervy LOF mutants (fig. S4, A to D; table S1), demonstrating that these phenotypes resulted from a lack of neuronal nervy. nervy LOF mutant phenotypes were qualitatively and quantitatively similar to those phenotypes observed following increased expression in all neurons [gain of function (GOF)] of PlexA or its downstream signaling partner MICAL (Fig. 2F; Fig. 3, B and H; table S1) (10, 11), which suggests that nervy may antagonize Sema-1a-PlexA repulsive axon guidance.

Nervy and type II PKA regulate Sema-1a-PlexA repulsion. (A) Percentage of defective ISNb, SNa, and CNS pathways per total number of hemisegments in wild-type embryos; nervy, pkaRII, Sema1a, PlexA, and MICAL LOF mutant embryos; and neuronal nervy, MICAL, and nervyV523P GOF mutant embryos. (B) Percentage of total defects shown in (A) exhibiting a decrease (top) or an increase (bottom) in repulsion (defined in table S1). (C to G) Filleted embryos as in Fig. 2. (C) Low muscle expression of Sema-1a using one copy of the GAL4 driver 24B and one copy of the transgene UAS-Sema1a results in wild-type motor projections. (D to G) Motor axon phenotypes are dramatically enhanced when embryos expressing low levels of Sema-1a in muscles as in (C) (UAS-Sema1a/+; 24B-GAL4/+) are also heterozygous for nervy or type II PKA. Loss of one copy of nervy (nervy/+) enhances Sema-1a–mediated repulsion and results in a failure of ISNb axons to defasciculate and innervate muscles 6/7 and 12/13 (arrows) (D) and of SNa axons to make their two characteristic turns (arrows) (E). Summarized in (G). (F) Percentage of defective ISNb and SNa pathways per total hemisegments in embryos expressing low levels of muscle Sema-1a (Muscle Sema-1a: UAS-Sema1a/+; 24B-GAL4/+), low levels of muscle Sema-1a, and also heterozygous for nervy (Muscle Sema-1a/nervy LOF: UAS-Sema1a/nervy PDFKG38; 24B-GAL4/+); or expressing low levels of muscle Sema-1a and also heterozygous for type II PKA (Muscle Sema-1a/pkaRII LOF: UAS-Sema1a/pka RIIEP(2)2162; 24B-GAL4/+). (H) Modelfor nervy-PKA regulation of semaphorin repulsion. Normally, Sema-1a–mediated repulsion (minus sign) drives selective axon-axon defasciculation, enabling target innervation. Decreasing this repulsion results in the failure of axonal defasciculation (left); increasing this repulsion directs increased and premature repulsion (right). Increasing nervy-PKA expression (increased cAMP) decreases Sema-1a–mediated repulsion such that the axon response to increasing cAMP is the same as when Sema-1a is decreased, and vice versa.

In contrast to nervy LOF phenotypes, overexpression of nervy in all neurons in a wild-type background (nervy GOF) decreased the ability of motor axons to defasciculate and innervate their muscle targets (Fig. 2, G and I; Fig. 3, A and B; fig. S3C; table S1). These phenotypes were consistent with the absence of, or inability to respond to, an axonal repellent and are identical to those seen in Sema1a, PlexA, MICAL, and Off-track (OTK, part of the Sema-1a signaling cascade) LOF mutants (Fig. 2I; Fig. 3, B and H; table S1) (10–13). ISNb axons often failed to defasciculate from each other, or even from the ISN, in nervy GOF mutants and bypassed their muscle targets (Fig. 2, G and I; Fig. 3, A and B; table S1). Likewise, axons within the SNa pathway failed to defasciculate in nervy GOF mutants and often stalled along their trajectory (Fig. 2I; Fig. 3, A and B; fig. S3C; table S1). Sema1a, PlexA, MICAL, and OTK LOF-like phenotypes (10–13) were also seen in the CNS of nervy GOF mutants: The outermost 1D4-positive longitudinal connective was thinner in some segments, discontinuous in others, and often fused with the middle 1D4-positive fascicle (Fig. 2, H and I; Fig. 3, A and B; table S1). These results support a role for nervy in antagonizing Sema-1a-PlexA repulsive axon guidance.

If nervy antagonizes Sema-1a signaling, then reducing nervy expression should increase the repulsive effects of Sema-1a. Very few axon guidance defects were observed when low levels of Sema1a were expressed in all muscles (Fig. 3, C and F; table S1) (11, 12). Expression of low levels of Sema-1a in all muscles in a nervy heterozygous background, however, resulted in an increase in Sema-1a repulsion and led to the inability of motor axons to defasciculate and innervate their muscle targets (Fig. 3, D to G; table S1). These phenotypes were identical to those seen when high levels of Sema-1a were expressed in all muscles (11, 12). This dominant enhancement of a weak Sema1a GOF phenotype by nervy, together with dominant suppression of a weak Sema1a LOF phenotype by nervy (fig. S5, A to E; table S1), suggest that nervy and Sema1a function in the same signaling pathway but have opposing effects, and that nervy acts downstream of Sema-1a to regulate repulsive guidance.

To test the necessity of nervy–type II PKA interactions in regulating Sema-1a-PlexA signaling, we made a single amino acid substitution of a proline for a valine residue in nervy (nervyV523P) that was analogous to a mutation that disrupts MTG16-PKA RII interactions (Fig. 1B) (4). We generated transgenic flies expressing epitope (myc)–tagged nervyV523P, but unlike neuronal expression of wild-type nervy in a nervy LOF mutant background, neuronal nervyV523P failed to rescue the nervy LOF mutant phenotypes (table S1). Therefore, we reasoned that nervyV523P might function in a dominant-negative manner by retaining its ability to bind to PlexA (fig. S1, B and C) but blocking the coupling of PKA to PlexA. Indeed, expression of myc-nervyV523P in all neurons in a wild-type background resulted in axon guidance phenotypes (Fig. 2, J to L; Fig. 3, A and B; fig. S3D; table S1) similar to those seen in nervy or pka RII LOF mutants (Fig. 2, D to F; Fig. 3, A and B; fig. S3B; fig. S4, E to H; table S1). These phenotypes are the opposite of those seen when wild-type nervy is expressed in all neurons and are indicative of increased Sema-1a-PlexA repulsion because they resemble MICAL and PlexA GOF mutants (Fig. 2F; Fig. 3, A and B; table S1). These results suggest that nervy's ability to bind type II PKA is critical for the modulation of Sema-1a-PlexA repulsive guidance.

We provide here evidence for cAMP-PKA modulation of Sema-1a–mediated repulsive guidance by the association of the AKAP nervy with the PlexA receptor (Fig. 3H; fig. S5L). Our results are consistent with in vitro observations describing a role for cAMP in regulating semaphorin repulsion in vertebrates (6, 8, 9). cAMP antagonists neutralize the effects of cyclic guanosine monophosphate (cGMP) on Sema3A repulsion (6), while Sema3A repulsion is increased by inhibiting PKA and neutralized by increasing cAMP (8, 9). The conserved nature of semaphorin-plexin signaling suggests that vertebrate MTG proteins (15) may underlie these observations as well as observations that increased cAMP levels enhance axonal regeneration in the mammalian CNS (16). Our observed interactions between plexin and nervy are also consistent with roles described for AKAPs in other signaling pathways, allowing precisely localized PKA to be maintained close to its activators and substrates (5). What activates PKA bound to nervy? Anchored PKA is poorly dissociated by basal cAMP levels (5), so inputs from other signaling pathways, such as those activated by netrin or G protein–coupled receptors, may specifically regulate semaphorin signaling by locally increasing cAMP and activating PKA (9, 17, 18). PKA targets in the vicinity of PlexA may include L-type Ca2+ channels (19), MICAL (10), or the Rho guanosine triphosphatase (1, 8). Therefore, modulation of semaphorin signaling through the direct coupling of PKA to plexin by an AKAP allows for the integration of multiple guidance cues by the neuronal growth cone, enabling local, rapid, and directed responses to a complex extracellular environment.